In a healthy individual, the immune system uses several different immune tolerance mechanisms in order to prevent the development of autoimmune disease. For T cells, these include thymic and peripheral mechanisms that result in elimination of autoreactive cells, such as receptor editing and deletion, as well as other mechanisms that allow T cells with potentially autoreactive T cell receptors (TCR) to remain as part of the TCR repertoire but to be controlled in their ability to respond. The latter include inhibitory mechanisms that act intrinsically to the cell, generally referred to as anergy; and mechanisms that regulate extrinsically, through the activity of regulatory T cells. Little is known about the underlying mechanism responsible for whether a self-reactive CD8 T cell in the periphery undergoes tolerance through deletion or anergy. Studies in this proposal will delineate the molecular pathways through which peripheral tolerance of nave CD8 cells occurs. Aim 1 will test the hypothesis that T cell receptor affinity determines whether tolerance occurs through anergy or deletion. The affinity of CD8 T cell activation will be varied using well-defined altered peptide ligands. We will also determine whether the tolerance mechanism induced through T cell recognition of antigen on cross-presenting dendritic cells in vivo depends upon the strength of TCR signaling. Preliminary data obtained through microarray analysis of gene expression in cells undergoing deletion and anergy has revealed a number of transcriptional features unique to each tolerance mechanism that form the basis for Aims 2 and 3 in this proposal. Aim 2 will examine the molecular pathways that are responsible for T cell deletion. We will test the hypothesis that deletion occurs as the result of induction of the tumor suppressors genes, GADD45g and FHIT. These may contribute to activation of the proapoptotic Bim through stress induced MAP kinases. This will be tested by inactivating the components of these pathways through the use of genetically deficient mice and pharmacological inhibitors. Aim 3 will test the hypothesis that the mechanism of anergy that occurs when nave CD8 cells are activated with a high dose of toleragen involves enhanced expression of one or more negative costimulatory molecules, including; PD-1, NKG2a and Semaphorin 7A.